This invention relates to bisulfite adducts of arginine aldehydes which are thrombin inhibitors and anticoagulants in mammals. In particular it relates to bisulfite adducts of L-Arginine aldehyde derivatives having high anticoagulant activity, antithrombotic activity, and oral bioavailability.
The process of blood coagulation, thrombosis, is triggered by a complex proteolytic cascade leading to the formation of thrombin. Thrombin proteolytically removes activation peptides from the A.alpha.-chains and the B.beta.-chains of fibrinogen, which is soluble in blood plasma, initiating insoluble fibrin formation.
Anticoagulation is currently achieved by the administration of heparins and coumarins.
Parenteral pharmacological control of coagulation and thrombosis is based on inhibition of thrombin through the use of heparins. Heparins act indirectly on thrombin by accelerating the inhibitory effect of endogenous antithrombin III (the main physiological inhibitor of thrombin). Because antithrombin III levels vary in plasma and because surface-bound thrombin seems resistant to this indirect mechanism, heparins can be an ineffective treatment. Because coagulation assays are believed to be associated with efficacy and with safety, heparin levels must be monitored with coagulation assays (particularly the activated partial thromboplastin time (APTT) assay). Coumarins impede the generation of thrombin by blocking the posttranslational gamma-carboxylation in the synthesis of prothrombin and other proteins of this type. Because of their mechanism of action, the effect of coumarins can only develop slowly, 6-24 hours after administration. Further, they are not selective anticoagulants. Coumarins also require monitoring with coagulation assays (particularly the prothrombin time (PT) assay).
Recently, interest in small synthetic peptides that are recognized by proteolytic enzymes in a manner similar to that of natural substrates has grown. Tripeptide aldehydes such as D-Phe-Pro-Arg-H, Boc-D-Phe-Pro-Arg-H, and D-MePhe-Pro-Arg-H, Bajusz et al., J. Med. Chem., 33, 1729-1735 (1990) demonstrate potent direct inhibition of thrombin. Many investigators have synthesized analogs in an effort to develop pharmaceutical agents, for example Shuman et al., J. Med. Chem., 36, 314-319 (1993). These small synthetic peptide derivatives contain an aldehyde group bonded to the arginine residue.
Although the heparins and coumarins are effective anticoagulants, and no drug has yet emerged from the known tripeptide aldehydes, and despite the continuing promise for this class of compounds, there exists a need for anticoagulants that act selectively on thrombin, and independent of antithrombin III, exert inhibitory action shortly after administration, preferably by an oral route, and do not interfere with lysis of blood clots, as required to maintain hemostasis.
Epimerization at the amino acid alpha-carbon is a known problem that plagues polypeptide synthesis and stability. This configurational inversion (loss of chiral integrity at one chiral center in a peptide having two or more chiral centers) may limit the prophylactic and therapeutic use of arginine aldehyde thrombin inhibitors. The aldehyde group on the arginine residue epimerizes from the preferred L configuration to the D diastereomer. Such configurational inversion occurs through a simple enolization reaction, cyclization (lactam) formation, or a combination of those reactions. These two reactions are influenced at least by the nature of substituents associated with the amino, carbonyl and alpha-carbon atom, temperature and basicity of the preparation, purification and reconstitution media.
Protection of the arginine aldehyde group from configurational inversion would greatly enhance the prophylatic and therapeutic efficacy of small synthetic peptides containing said group as thrombin inhibitors as epimerization to the D diastereomer is believed to inactivate the molecule as a thrombin inhibitor.
The present invention is directed to the discovery that the compounds of the present invention, as defined below, are potent thrombin inhibitors that have high bioavailability following oral administration; and importantly are substantially stabilized to configurational inversion.
Accordingly, it is a primary object of the present invention to provide configurational inversion stabilized bisulfite adducts of L-arginine aldehyde derivatives that are potent thrombin inhibitors useful as anticoagulants.
Other objects, features and advantages will be apparent to those skilled in the art from the following description and claims.